Magnetic pin for concentrating and separating particles

ABSTRACT

The invention relates to a magnetic pin ( 8, 22 ) for concentrating particles, containing 
     (a) a grip adapter ( 2, 14 ), 
     (b) a connecting pin ( 4, 16 ) with dents ( 3, 15 ), 
     (c) at least one magnet ( 7, 20 ) and 
     (d) a sheath ( 5, 18 ).

The present invention relates to a magnetic pin for concentrating andseparating particles.

The latest detection methods for nucleic acids, such as polymerase chainreaction (PCR), ligase chain reaction (LCR), nucleic acid sequence basedamplification (NASBA), strand displacement amplification (SDA), can becarried out by amplification of nucleic acids with extreme sensitivity.Because of this sensitivity the methods are very susceptible tocontaminations. In this connection, the danger of contaminationincreases with the increasing number of steps and the manipulations tobe made, such as centrifugation and pipetting steps. Thus, it should beimportant to the experimenters to keep the number of steps andmanipulations as small as possible. However, this is often problematic,since differing sample volumes must be observed for differing steps andconcentration of the sample volume is necessary in between. Therefore,there is a demand for a possibility of dispensing with manipulationsteps, particularly centrifugation steps, in the above mentioned method,by taking the sample to be analyzed directly out of the various vessels,such as cups or microtiter trays, and transferring it. This demand ofeasily transferring samples from a large volume to a smaller volume andbeing able to easily change the sample containers, respectively, is, ofcourse, not only limited to the field of nucleic acid technique butexists throughout (bio)chemistry where the concentration of samples isnecessary.

Therefore, it is the object of the present invention to provide anapparatus by means of which target molecules can easily be taken out ofthe containers or vessels and transferred into other vessels so as to beable to dispense with manipulation steps, such as centrifugation steps,and work with small volumes.

This object is achieved by a magnetic pin according to claim 1.Advantageous embodiments follow from the subclaims.

In order to derive an advantage for the above mentioned methods by meansof the magnetic pin according to the invention, magnetic labeling of thetarget molecules must, of course, be given. A person skilled in the artknows how to achieve this. For example, this is achieved in that thetarget molecules as such have magnetic properties or are bound to“magnetic beads”. If a detection method is concerned, anotherpossibility is to link streptavidin to iron so as to bind the targetmolecules which in turn are labeled with biotin.

In an exemplary method in which the magnetic pin according to theinvention is used, the sample material, e.g. serum, plasma, whole blood,cells or tissue, is subjected to a chemical or chemico-enzymaticbreaking-up and a denaturation, respectively. This solutionsimultaneously contains a first nucleic acid oligonucleotide (primer)which hybridizes as a probe to the target molecules. Thisoligonucleotide is covalently bonded to a biotin molecule which can bebound by streptavidin with high affinity. This streptavidin in turn isbound to particles which contain iron. Because the streptavidinparticles cannot be added to a denatured sample mixture, since otherwisethey would denature as well and would lose their bonding properties tobiotin, the sample mixture must be diluted, which increases the samplevolume. However, this great sample volume has a negative effect onsubsequent steps, such as amplifications. For example, the resultingconjugates (sample—oligonucleotide—biotin—streptavidin—iron) aretransferred by means of the magnetic pin according to the invention intoanother container where the further steps can then take place in a muchsmaller volume.

The invention is further described by means of the figures in which:

FIG. 1a shows individual components of a magnetic pin 8 according to theinvention,

FIG. 1b shows the assembled magnetic pin 8 according to the invention,

FIG. 2 shows the transfer of particles from a large sample containerinto a small sample container,

FIG. 3a shows individual components of another magnetic pin 22 accordingto the invention,

FIG. 3b shows the assembled magnetic pin 22 according to the invention.

The individual components of an embodiment of a magnetic pin accordingto the invention are shown in FIG. 1a. The magnetic pin 1 has a gripadapter 2 at its rear end. It is developed such that it can be grippedand operated by an industrial automatic pipetting machine. Here, thegrip adapter 2 is preferably made of a deformable material, such asrubber. In addition, the magnetic pin 1 comprises a connecting pin 4having a dent 3 so as to form a grip for the automatic pipettingmachine. Furthermore, the dent serves for holding a sheath 5. Thissheath 5 is thin-walled (wall thickness about 0.2 to 0.7 mm, preferably0.3 mm) and consists preferably of polymeric plastics (e.g.polypropylene) and a non-magnetizable, but conductive, material,respectively, e.g. plastics having a graphite portion. The sheath 5 haspreferably a conical shape at its lower end and has a constriction 6,which is preferably adapted to sample containers such that with a smallliquid volume (e.g. 25 μl in a PCR cup) it is just wet in a filledsample container. The connecting pin 4 should be made of a non-magneticmaterial, such as aluminum. The front end of the magnetic pin has amagnetic head 7 which has preferably a conical shape. Every conventionalpermanent magnet is in consideration for this.

FIG. 1b shows a magnetic pin 1 provided with the sheath 5 as themagnetic pin 8 according to the invention.

The concentration of the magnetically labeled sample material present inthe sample containers is preferably carried out with the magnetic pin 8disposed in the sheath and preferably proceeds as shown in FIG. 2.

I) The magnetic pin with the sheath 8 is immersed in a large samplecontainer 9 including magnetically labeled particles 10 disposed in aliquid. Pin and sheath are slowly moved downwards. The particles areattracted by the magnetic head of the magnetic pin.

II) The magnetic pin with sheath 8 is slowly drawn out of the samplecontainer again, so that only minor liquid residues adhere to thesheath. The adhering particles can optionally be subjected to furthertreatments in a wash vessel without a loss of the adhering particlesoccurring.

III) The tip of the magnetic pin including the sheath surrounding it areimmersed in a smaller sample container 11. Because of the adaptation ofthe magnetic head 7 including the surrounding sheath to the shape of thesample container, only a small liquid volume is required to wet thesheath of the entire tip. The sheath is fixed in stand or support 12.

IV) The magnetic pin 1 is drawn out of the sheath by a rapid movement.The particles separate, in the model case, from the sheath because ofthe omitted magnetic force and stay in the sample container 11. Shouldthis not suffice, they will be pulled downwards by another magnetlocated externally underneath the sample container.

V) The sheath 5 is carefully removed from the sample container 11.

Result: The particles are now found in a smaller volume in anothersample container.

Another preferred embodiment of a magnetic pin according to theinvention is shown in FIG. 3. The individual components of such amagnetic pin are shown in FIG. 3a. As in FIG. 1, the magnetic pincomprises a grip adapter 14, a dent 15, a connecting pin 16, a sheath 18and a constriction 19. The above-mentioned product and dimensionalparameters for these components also apply to the embodiment accordingto FIG. 3. In the embodiment shown in FIG. 3 the magnetic forces, whichshall escape the tip 17 in beamed fashion, emanate from a relativelygreat permanent magnet 20 which is mounted farther above, and aredirected e.g. by an iron core to the tip 17. In this case, the magneticfield directly emanating from the magnet can be shielded by a metallicsheath 21, such as an iron sheath. The magnet 20 can be disposed at anyheight on the connecting pin 16. However, it is preferably found nearthe grip adapter, since it can have a disturbing effect if locatedfarther downwards when the magnetic pin is immersed in a samplecontainer. The tip 17 consists of a magnetizable material and can havemagnetic properties itself, respectively, i.e. can be the continuationof the iron core.

FIG. 3b shows a magnetic pin 13 provided with a sheath 18 as themagnetic pin 22 according to the invention.

According to the invention, the magnetic force shall effect theseparation of target substances through the help of other suitablesubstances. Therefore, it is not the magnet as such that is decisive butits shape and its arrangement. The shape of the magnet influencesdecisively the course of the magnetic field and thus its force, on theone hand, and the shaping must be accurately in conformity with thetarget application in this case, on the other hand. In a preferredembodiment, the collection of magnetic particles from solutions (such asserum) takes place on the smallest possible area (tip of the magneticpin). In this case, the magnetic force shall be as high as possible, sothat the magnetic particles can be collected over a suitable containercross-section. Additional collection of the magnetic particles from asubstantially greater starting volume becomes possible by slowlylowering the magnetic pin within the container (e.g. serum tube). Inaddition, the shape of the magnetic tip must be adapted to the fact thatwhen the magnetic pin is withdrawn the least possible liquid adheresthereto. At the same time, it must be taken into consideration inconnection with the tip shape that when the pin is immersed insolutions, not even minute parts of this solution (droplets, aerosols)may pass into the air. In the final analysis, the shape of the pin mustalso be adapted to the target vessel, so that it suffices to supply theleast possible liquid volume there to fully wet the site of the adheringmagnetic particles. The object of the plastic sheath of the magneticpin, which is part of the patent, is at this time to enable the stoppingof the magnetic force. In order to achieve this objective, the magneticpin is smartly drawn out of the sheath, whereupon the magnetic particlesfall off the outside of the sheath.

Thus, the embodiment according to the present invention, which has apointed shape of the magnet, offers special advantages when withdrawnfrom a solution. This process can also be automatically controlledtemporally via a measurement of the induction when conductive sheathsare used, such that no drop is left at the tip withdrawn from the liquidsurface. The starting solution has maximum reduction (single-stepconcentration). This is an important detail not only for the preventionof contamination dangers while moving over other samples but also forthe separation and concentration of magnetic particles which is notpossible in this way when a flat tip is used. Furthermore, the magneticfield has transverse orientation when the magnet has a pointed shape, sothat an improved collective effect occurs.

As a result, the magnetic particles can be supplied in an extremelysmall liquid volume by a pointed shape which has to be adapted to theemployed containers. If a corresponding vessel is used on the receivingside, a magnetic tip can also be used for transferring substances andremoving impurities by washing from a very small starting volume.

Another advantage of the magnetic pin according to the invention is thatthe target molecules of the sample are easily concentrated andseparated. Furthermore, this is inexpensive, since the sheath is made ofcheap material and can be discarded and rinsed, respectively, whereasthe actual magnetic pin has a long service life and can be used againwithout further purification, since in the model case it is not incontact with the sample.

What is claimed is:
 1. Magnetic pin (8, 22) for concentrating particles,comprising: a connecting pin (4, 16) comprising a rear end and a frontend (7, 17) and being provided with a dent (3, 15); at least one magnet(7, 20) which is arranged at the connecting pin (4, 16), such that amagnetic filed emanates in concentrated fashion at the front end (7,17); and a sheath (5, 18) into which the connecting pin (4, 16) can beintroduced, which has a conical shape at its lower end, and consists ofpolymeric plastics and a non-magnetizable, conductive material; whereina grip adapter (2, 14) is arranged which serves for operating themagnetic pin (8, 22) through an automatic pipetting machine; the frontend of the connecting pin (4, 16) is provided with a magnetic head (7)which has a conical shape; and the sheath is moved in associated manneror separately from the connecting pin.
 2. The magnetic pin according toclaim 1, wherein the grip adapter (2, 14) is made of a deformablematerial.
 3. The magnetic pin according to claim 1 or 2, wherein thegrip adapter (2, 14) is developed such that it can be gripped andoperated by an industrial automatic pipetting machine.
 4. The magneticpin according to claim 1 or 2, wherein the connecting pin (4, 16) ismade of a nonmagnetic material.
 5. The magnetic pin according to claim 1or 2, wherein the magnet (7, 20) is a permanent magnet.
 6. The magneticpin according to claim 1 or 2, wherein the sheath (5, 18) is made of anonmagnetizable material.
 7. The magnetic pin according to claim 1 or 2,wherein the sheath (5, 18) is made of polypropylene.
 8. The magnetic pinaccording to claim 1 or 2, wherein the sheath (5, 18) has a wallthickness of 0.2 to 0.7 mm.
 9. The magnetic pin according to claim 1 or2, wherein the dent (3, 15) has a shape adapted for fixing the sheath(5, 18).
 10. The magnetic pin according to claim 1, wherein theconnecting pin at the front end comprises the at least one magnet asmagnetic head.
 11. The magnetic pin according to claim 1, wherein thefront end is a tip and the connecting pin has a magnet whose magneticforces emanate from the tip in beamed fashion.